Chromatin alterations imposed by the oncogenic transcription

139 Pages·2007·6.23 MB·English

Universitat Pompeu Fabra
Facultad de Ciencias de la Salud y de la Vida

Chromatin alterations imposed by the
oncogenic transcription factor PML-RARα

Lluís Morey Ramonell
PhD thesis
Barcelona, February 2008

Carrer Corretger, num.14, 1er 3a
C.P 08003
e-mail: [email protected]
telf. 649 98 99 96

Universitat Pompeu Fabra
Facultad de Ciencias de la Salud y de la Vida

Chromatin alterations imposed by the
oncogenic transcription factor PML-RARα

Memoria presentada por Lluís Morey Ramonell para optar al grado de Doctor.
Este trabajo ha sido realizado bajo la dirección del Dr. Luciano Di Croce en el
Programa de Diferenciación y Cáncer del Centro de Regulación Genómica
(CRG) de Barcelona.

Dr. Luciano Di Croce

Table of contents
Table of contents
SUMMARY 1

INTRODUCTION 5
1. Leukemia. 7
1.1 - Leukemia. 7
1.2 - Acute Promyelocytic Leukemia (APL) and PML-RARα. 8
1.3 - APL treatment. 9
1.4 - Retinoic Acid Receptor α (RARα) 10
1.5 - Promyelocytic Leukemia protein (PML) 12
1.6 - PML-RARα-mediated repression. 14

2. Epigenetics. 16
2.1 - Chromatin structure and the “Histone Code”. 16

3. DNA methyltransferases and DNA methylation. 18
3.1 - DNA methylation. 18
3.2 - DNAmethyltransferases (DNMTs) 19
3.3 - Pattern of DNA methylation during development. 21

4. Chromatin remodeling. 22
4.1 - Nucleosome Remodelling and Deacetylase complex (NuRD) 24
4.2 - The NuRD complex in gene regulation. 25
4.3 - Role of the NuRD complex in development. 26
4.4 - Function of the subunits of NuRD complex. 28
4.4.1 – Histone Deacetylases proteins (HDACs) 28
4.4.1.1 - Class I 29
4.4.1.2 - Class II 30
4.4.1.3 - Class III 30
4.4.2 – Methyl-CpG-binding domain proteins (MBDs) 31
4.4.2.1 - MBD3. 32
4.4.3 – Metastasis associated proteins (MTAs) 33
4.4.4 - Chromodomian CHD3-CHD4 ATPase
sub-familiy (CHDs) 35
4.4.5 - Retinoblastoma Associated Proteins 46/48
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(RbAps) and p66 37

5. Polycomb group of proteins (PcG). 37
5.1 - PcG protein complexes. 38
5.2 - Transcriptional repression by PRCs. 39
5.3 - PcGs and cancer. 40

OBJECTIVES 43

RESULTS 47
1. PML-RARα expression induces chromatin alteration. 50
1.1 - PML-RARα and histone H1 are recruited to RARβ2 promoter
while there is a concominantly replacement of histone H3.3. 51

2. PML-RARα interacts with NuRD. 52
2.1 - PML-RARα interacts with overexpressed NuRD subunits
in 293T cells. 53
2.2 - PML-RARα and NuRD complex interact directly through
both PML and RARα moieties. 55
2.3 - Endogenous interaction of PML-RARα and the NuRD
complex in U937-PR9 and leukemic-NB4 cells. 56

3. NuRD occupancy at PML-RARα target promoters. 58
3.1 - PML-RARα recruits the NuRD complex to its target
promoters. 58
3.2 - The NuRD complex is released from PML-RARα-
target genes, upon RA administration, in leukemic cells. 60

4. The NuRD complex is required for structural changes of
PML-RARα-target promoters. 63
4.1 - The NuRD complex is necessary for efficient repression of
RARβ2-target PML-RARα gene. 64
4.2 - The NuRD complex facilitates PML-RARα-induced structural 65
changes on its target promoter.
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5. The NuRD complex facilitates DNA hypermethylation in
U937-PR9 cells. 67

6. Role of the NuRD complex in the crosstalk between DNA and
histone methylation. 73

7. Epigenetic changes in NuRD knockdown leukemic cells. 76

8. The NuRD complex is involved in the maintenance of leukemia. 80

DISCUSSION 83
1. PML-RARα and NuRD. 85
2. Link NuRD and DNA methylation. 86
3. Histone Deacetylation. 88
4. NuRD and PRC2. 89
5. NuRD and cell differentiation. 90

CONCLUSIONS 93

MATERIALS AND METHODS 97
1. Plasmids. 99
2. Antibodies. 99
3. Cell lines. 100
4. Calcium phosphate transfections. 100
5. Immunoprecipitation and Western-Blot analysis. 101
6. Chromatin immunoprecipitation. 101
7. GST-fusion, IVT and pull down assays. 102
8. RNAi and retroviral infection. 103
9. DNase hypersensitive site assay. 104
10. Bisulfite genomic sequencing. 105
11. Differentiation assays. 105
12. RNA purification and RT-PCR analysis. 106
13. DNAmethyltransferase assay. 107
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14. Primer sequences table. 107

BIBLIOGRAPHY 109

PUBLICATIONS 122
Summary